Aminomethyl-Derived Beta Secretase (BACE1

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Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality Christopher R. Butler, Kevin Ogilvie, Luis A Martinez-Alsina, Gabriela Barreiro, Elizabeth M. Beck, Charles E Nolan, Kevin Atchison, Eric S Benvenuti, Leanne M Buzon, Shawn D Doran, Cathleen Gonzales, Christopher J. Helal, Xinjun Hou, Mei-Hui Hsu, Eric F. Johnson, Kimberly Lapham, Lorraine F Lanyon, Kevin Parris, Brian T. O'Neill, David R. Riddell, Ashley Robshaw, Felix F. Vajdos, and Michael A. Brodney J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01451 • Publication Date (Web): 06 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Journal of Medicinal Chemistry

Aminomethyl-Derived Beta Secretase (BACE1) Inhibitors: Engaging Gly230 without an Anilide Functionality Christopher R. Butler*a, Kevin Ogilviec, Luis Martinez-Alsinac, Gabriela Barreiroa, Elizabeth M. Becka, Charles E. Nolanb, Kevin Atchisonb, Eric Benvenutie, Leanne Buzonc, Shawn Dorane, Cathleen Gonzalesb, Christopher J. Helalc, Xinjun Houa, Mei-Hui Hsuf, Eric F. Johnsonf, Kimberly Laphame, Lorraine Lanyone, Kevin Parrisd, Brian T. O’Neillc, David Riddellb, Ashley Robshawb, Felix Vajdosd, and Michael A. Brodneya a

Neuroscience & Pain Medicinal Chemistry, bNeuroscience & Pain Research Unit, Pfizer Worldwide

Research and Development, 610 Main St, Cambridge, MA, 02139, United States; cNeuroscience & Pain Medicinal Chemistry, dCenter of Chemistry Innovation and Excellence, ePharmacokinetics, Dynamics, and Metabolism, Pfizer Worldwide Research and Development, 445 Eastern Point Road, Groton, CT, 06340, United States, fMolecular and Experimental Medicine, The Scripps Research Institute, 10550 Torrey Pines Road, La Jolla, California, 92024, United States

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required ABSTRACT A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site

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leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogs with an excellent balance of ADME properties and potency; however, potential drug-drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor. Introduction Alzheimer’s disease (AD), a neurological disorder that imparts a slow progression of cognitive decline, dementia, and ultimately death, has yet to yield to a significant enhancement in treatment or prevention. Disease progression is marked by the deposition of amyloid β (Aβ)derived plaques in the hippocampal and cortical regions of the brain. The amyloid hypothesis proposes that increased Aβ production or its decreased clearance is responsible for the molecular cascade that eventually leads to neurodegeneration and AD.1,2 Aβ production is initiated by the proteolytic cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme (BACE1) within the endosome,3 to afford a soluble N-terminal ectodomain of APP (sAPPβ) and the C-terminal fragment C99.4 The membrane-bound C99 is then cleaved by γ-secretase to release Aβ, including Aβx-40 and Aβx-42 isoforms.5 Recently, an APP “loss of function” mutation, with protective effects against AD, has been reported to be cleaved more slowly by BACE1.6 Modulation of the Aβ cascade via safe and effective inhibition of BACE1 has remained a target of great interest for a number of years.7 Considering the chronic dosing regimen required for a successful AD treatment, an exquisitely selective and safe profile for a BACE1 inhibitor is paramount. Of particular concern


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for this target is inhibition of hERG,8 as well as related aspartyl proteases including Cathepsin D (CatD), which has confounded early generations of BACE1 inhibitors.9 The hERG-mediated cardiovascular liability is traditionally avoided by eliminating basic amine functionality and lowering lipophilicity.10 This is challenging for BACE1 as the active site is most efficiently engaged through utilization of such an amine, thus requiring alternate mitigation strategies. Additionally, the binding sites of CatD and BACE1 have high sequence similarity, and therefore differentiation requires exploitation of subtle architectural variances in order to maintain affinity for BACE1 while avoiding CatD inhibition. Compounds that fail to achieve sufficient selectivity over CatD carry a liability for ocular toxicity, due to the resulting accumulation of fluorescent material in the retinal pigment epithelium (RPE) layer.9 The physiological relevance of BACE2 has emerged in recent years, first as an enzyme involved in pigmentation processing, specifically acting on PMEL17 in the periphery.11 Improper functioning of BACE2 is believed to result in hypopigmentation.12 BACE2 is also expressed in the pancreas and plays a role in glucose homeostasis. To our knowledge, there are limited examples of BACE1 inhibitors possessing significant selectivity over BACE2. Compounds that lack this selectivity window and exhibit impaired access to the brain will therefore inherently suffer from significant inhibition of BACE2. In summary, agents developed for chronic BACE1 inhibition should be designed to minimize activity against related proteases such as CatD and BACE2. The amidine-containing BACE1 inhibitors, reported by a number of groups, provide a suitable scaffold to systematically address the CatD and hERG liabilities.13 A number of these inhibitors, such as MK-8931 (1), have recently entered clinical studies; two of them are shown in Figure 1.14 A common construct within this class is an amide moiety connecting two aromatic



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rings that ultimately occupy the S1/S3 pockets when bound in the BACE1 active site. The incorporation of this moiety generally confers potent inhibition of BACE1 in addition to exquisite selectivity over CatD. Unfortunately, these merits are generally offset by increased Pgp-mediated efflux, resulting in decreased brain penetration. There is a correlation between the presence of a third hydrogen bond donor (HBD) and an increased likelihood of efflux transporter liabilities.15 Poor brain penetration inherently increases the body burden required to achieve the desired brain concentrations, and thus further exacerbates any issues arising from less than exquisite aspartyl protease selectivity. Moreover, recent reports have shown that there are relevant peripheral substrates for BACE1 in addition to the targeted central APP processing.16

Figure 1. Selected literature BACE1 inhibitors Inhibitors bearing the P1/P3 amide motif not only exhibit higher efflux transporter liability, but also contain a metabolic soft spot associated with amidase activity, which in this case would reveal anilines upon amide cleavage.17

In addition to this potential metabolic

liability, anilines are themselves a structural alert, known to be a culprit for downstream toxicity associated either with oxidation of the electron-rich aryl ring and subsequent trapping with ambient nucleophiles, or oxidation of the nitrogen itself to the N-oxide.18 Design Criteria.


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Despite a significant and sustained effort, the identification of a potent, safe, selective BACE1 inhibitor with a balanced ADME profile, including good brain penetration, remains challenging. Our design criteria were therefore to identify an orally efficacious BACE1 inhibitor that: a) demonstrates excellent selectivity (>100x) over hERG, CatD and the related aspartyl proteases; and b) maintains good brain penetration (Cb,u/Cp,u > 0.5 as measured in rodents) without the use of an anilide functionality. We therefore sought to identify an amide replacement that provided the opportunity for similar efficiency gains while avoiding the potential for toxicity imparted by the buried aniline moiety. The recent BACE1 crystal structure of 2, published by Roche, provides a structural rationale for the high inhibitory efficiency of P1/P3 amides (Figure 2). 19 The amide N-H is positioned at a near-ideal distance and angle to engage the backbone carbonyl of the Gly230 residue in a hydrogen bonding interaction. Additionally, the coplanar conformation of the rings occupying the S1/S3 pockets serves to efficiently fill the shallow lipophilic binding site.



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Asp-228

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Tyr-71 flap

Gly-230

S1 S3

10s loop

Figure 2. Interaction between Gly230 and 2 (PDB: 3ZMG, add 61 to residue numbers to match 3ZMG’s number) Thus, a suitable amide replacement would need to fulfill these two structural requirements: effective engagement of Gly230 and reasonable concomitant occupation of the S1/S3 subpockets. Simple reversal of the amide removes the buried aniline while retaining the hydrogen bond donor, but rotation around the amide bond is then needed to recapitulate the optimum hydrogen bond angle, resulting in significant clashes with the edge of the S3 subpocket. In contrast, a homologated aminomethyl linker (Figure 3) addresses both of the aforementioned requirements. Among the salient features of this aminomethyl moiety, one key departure from the amide-derived analogs is its non-planarity. The sp3 nature of the benzylic carbon results in the two-atom unit preferring a nearly orthogonal orientation of the C-N bond relative to the


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fluorophenyl P1 ring, facilitating an optimal geometry for hydrogen bonding to Gly230. Optimization of the amine substituent in this alternative vector therefore becomes crucial, to balance the interplay of requisite hydrogen-bond donating ability of the amine with the resultant ADME characteristics of these compounds.

Tyr-71

Asp-228

flap

Gly-230

S3 S1

10s loop



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Figure 3. Strategy to mitigate challenges associated with amide pharmacophore of literature BACE inhibitors. Modeled alignment of aminomethyl design (orange) with compound 2 (yellow). Results and Discussion As it was not clear what structural attributes would be required to fill the S3 pocket in this new series, parallel (library) synthesis was employed to broadly evaluate structure-activity relationships for BACE1 inhibition. Compounds 6a-u were prepared by the three-step protocol illustrated in Scheme 1. Formylation of the previously described bromide 320 provided aldehyde 4, which could be converted to the corresponding protected amidines 5 via a reductive amination with the requisite amines. Removal of the amidine protecting group using standard conditions provided the analogs of interest. Scheme 1a

Reagents and conditions: (a) MeLi, Et2O, n-BuLi, then DMF, -78 oC, 92%; (b) amine, Na(OAc)3BH, DCE, rt, 22-97%; (c) HCl, dioxane, rt, 19-90%; (d) benzoic anhydride, TEA, THF/MeOH (2:1), rt, 86%; (e) N2H4·H2O, EtOH, rt, 31%. P = Boc or benzoyl

Analogs were evaluated in a panel of BACE1, BACE2, and CatD enzymatic assays (cellfree assay, CFA), as well as a whole-cell assay (WCA) reporting changes in soluble APP (sAPPβ) protein concentrations, indicative of APP processing by BACE1. The data from this assay panel, as well as key ADME parameters, for 6a–6k are provided in Table 1. The direct replacement of the amide linker with an aminomethylene spacer, as exemplified by 6a, yields


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weak activity at BACE1 (CFA IC50 = 73.5 µM), in stark contrast to the low nanomolar potency observed for many of the corresponding amides reported in the literature. In addition, the significant ADME challenges for this series are well illustrated by 6a, which exhibits a high MDR efflux ratio (Er) and significant clearance in human liver microsomes (HLM). Analogs 6b-d, which contain small alkyl substituents, show a modest improvement in BACE1 CFA potency, in concert with a dramatic increase in WCA potency (~1000x shifted relative to the CFA) and excellent selectivity over CatD. The improvement is most marked when considering ligand and lipophilic efficiencies (LE, LipE) for these low molecular weight, polar, dibasic amine analogs.

These compounds have significantly reduced clearance relative to 6a, as

measured by HLM, although they still exhibit significant P-gp transporter liability. Tertiary amines 6d-e are significantly less active in the BACE1 CFA but still exhibit good potency in the WCA, albeit an overall decrease in LipE, relative to the secondary amines. Ether-containing substituents are also well tolerated (6e,f), showing similar potency in the WCA, low microsomal clearance but with modest to high efflux ratios. Introduction of a branched methyl group (6c vs 6b and 6h vs 6f) offers an enhancement in potency (WCA) and a modest improvement in selectivity over BACE2. Tying this branching back into a 1,1,1-bicyclopentane (6i) is tolerated from a potency perspective, but negatively impacts the clearance, potentially due to the increased overall lipophilicity. Reduction in the pKa of the benzylic amine center (“pKa2”) was attractive due to the potential to simultaneously impact P-gp efflux and hERG liabilities. Indeed, addition of an electron-withdrawing trifluoromethyl group onto the amine significantly improves both potency and ADME balance. Although the tertiary amine (6j) is much less potent in the WCA, the secondary amine (6k) retains good WCA potency and comparable LipE to the more basic



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analogs, and exhibits enhanced BACE1 CFA potency and improved selectivity over BACE2 (5.8x, vs 3.2x for 6f). Although both CF3 analogs have significantly improved MDR-based efflux ratios (1.7 and 1.2), the secondary amine 6k shows greater metabolic stability (HLM CLint = 12.0 mL/min/kg) than the methylated version 6j (HLM = 29.4 mL/min/kg) Table 1. In Vitro Data for 6a-k

WCA LE/LipE

2.4

HLMf (mL/m in/ kg) 199

2.0

100

0.006 (1,366x)

3.9

-0.3

100

0.053 (1,186x)

9.0

-0.5

100

0.004 (1,045x)

6.8

-0.3

100

CatD CFA IC50 (µM) c >100

BACE1 WCA IC50 (µM)d

MDR Ere

LogD

1.35 (54x)

8.8

13.9

75.7

>100

0.008 (1,737x)

15.5

>94

>100

50.9

>100

40.8

6a

6b

6c

6d

6e

6f


0.34/ 5.2

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6j

20.2

>84

>100

1.013 (19.9x)

1.7

1.2

29.4

0.36/ 5.1

6k

1.31

7.6

>100

0.060 (21.8x)

1.2

1.0

12.0

0.45/ 6.7

a

IC50 values obtained from BACE1 CFA. bIC50 values obtained from BACE2 CFA. cIC50 values obtained from CatD CFA. dIC50 values obtained from BACE1 WCA; value in parentheses is the BACE1 CFA/WCA ratio. eRatio from the MS-based quantification of apical/basal and basal/apical transfer rates of a test compound at 2 μM across contiguous monolayers from MDR1-transfected MDCK cells. f Hepatic clearance predicted from in vitro human microsomal stability study.

It was clear that the additional basic amine exerts a profound effect on whole-cell potency, reinforced by comparison to the previously described monocyclic analog devoid of a P3 group (compound 6 in reference 21, BACE1 CFA IC50 = 36 µM, WCA IC50 = 636 nM).21 The low molecular weight and dibasic nature of the small alkyl exemplars result in highly ligand- and lipophilic efficient compounds in the whole-cell assay. While a similar rank order trend is observed for the BACE1 CFA, there is a 20- to 7,750-fold disconnect observed between the two assay formats. This disconnect can be rationalized by the presence of the second basic center increasing the propensity for accumulation into the endosome, an acidic intracellular compartment where BACE1 is thought to be localized,3 in the WCA format. As expected, decreasing the basicity of the amine by the installation of an electron-withdrawing group significantly compresses the CFA/WCA disconnect. It was recognized from the outset that the diversion from an amide moiety would likely result in a very different BACE1 substituent-derived SAR relative to the amides, as well as presenting distinct challenges to achieving a balanced ADME profile. A second round of amine optimization (Table 2) explored utilization of an electron-withdrawing group to balance ADME properties as well as incorporation of branched amines to further enhance potency. In an effort



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to more clearly define the impact of the EWGs on ADME balance, we measured the pKas for the amidine (“pKa1”) and benzylic amine (“pKa2”) for each of the analogs.22 Removal of one of the fluorines (6l) increases pKa2 relative to analog 6k, therefore increasing MDR Er (1.2 vs 3.1). Introduction of two geminal methyl groups provides a significant improvement in BACE1 CFA potency (6m vs 6k), albeit with a corresponding increase in MDR Er and a decrease in BACE2 selectivity (2x). Removal of one of the methyl groups (6n) decreases potency both in BACE1 CFA (1.9 µM) and WCA (64 nM), but improves efflux and BACE2 selectivity while reducing CYP 2D6 inhibition. Increasing the distance between the amine and trifluoromethyl groups (6o) increases pKa2 and diminishes potency in both assay formats. In an effort to capitalize on the potency increase associated with increased substitution, cyclopropylamine-containing analogs (6p-q) were prepared. The cyclopropane unit is well tolerated, as exhibited by the excellent WCA potency, but suffers from a significant degradation in BACE1 CFA potency and MDR Er. The addition of a difluoromethyl group (6r) improves the BACE1 CFA potency and reduces the increase in MDR Er, but simultaneously introduces a metabolic liability (HLM = 22 mL/min/kg). Replacement with a nitrile (6s) enhances BACE2 selectivity, but the MDR Er increases significantly (MDR Er = 6.0). Use of an oxetane (6t) in place of the cyclopropane improves WCA potency and decreases LogD, but results in an elevated MDR Er ratio (MDR Er = 3.5) nearly equivalent to that of the parent cyclopropylamine 6p. In contrast, the CF3-cyclopropyl derivative 6u maintains good potency in both assay formats, exhibits a minimal efflux ratio and low clearance, and maintains excellent selectivity over CatD (470x) and modest selectivity over BACE2 (5.9x). Table 2. In Vitro Data for Compounds 6k-6u


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Cpd

R=

BACE1 CFA IC50 (µM)a

BACE2 CFA IC50 (µM)b

CatD CFA IC50 (µM)c

MDR Ere

LogD

HLM (mL/ min /kg) f

CYP 2D6g

pKa1/ pKa2h

hERG (µM)i

>100

BACE 1 WCA IC50 (µM)d 0.060

1.31

7.60

1.2

1.0

12.0

25%

8.9/ 3.8

6.1

5.89

27.3

>100

0.058

3.1

0.3

13.0

21%

8.9/ 5.2

14.7

0.49

1.02

>100

0.031

3.6

1.5

100

0.064

2.2

0.6

11.9

45%

8.8/ 3.4

13.9

54.7

>100

>100

0.400

6.1

0.5

9.3

39%

8.9/ 6.0

20.4

20.6

>100

>100

0.030

3.4

0.5

100

0.014

6.9

0.1

100

WCA IC50 (nM)d

MDR Ere

0.006

2.3

LogD

1.1

HLMf (mL/ min /kg) 29

CYP 2D6 IC50 (µM)g 9.1

a

pKa1/ pKa2

7.91/ 3.80

hERG (µM)h

Cu,b/ Cu,p

Ceff, (Cu,b, nM)

4.3

0.25

31

IC50 values obtained from BACE1 CFA. bIC50 values obtained from BACE2 CFA. cIC50 values obtained from CatD CFA. dIC50 values obtained from BACE1 WCA. eRatio from the MS-based quantification of apical/basal and basal/apical transfer rates of a test compound at 2 μM across contiguous monolayers from MDR1-transfected MDCK cells. fHepatic clearance predicted from in vitro human microsomal stability study. gCYP 2D6 inhibition was obtained by measuring inhibition of 5 µM dextromorphan in pooled HLM (HL-MIX-102). hMeasured IC50 in hERG- expressing CHO cells.


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Figure 8. Effect of fused THP analog 16 on brain Aβx-42 following subcutaneous administration in wild-type mice. The observation of such disparate responses in the relative affinities to BACE1 and CYP 2D6 for 16 versus 6k was striking. The addition of the THP ring resulted in a 17x increase in potency at BACE1 while essentially having no impact on CYP 2D6 affinity (CYP 2D6 IC50 for 6k = 14 µM). A crystal structure of 16 in BACE1 was generated and confirmed that, as expected, reorganization of the S2’ subpocket in BACE1 had occurred, driven by the rotation of Tyr71 to accommodate the THP ring and fluoromethyl substituent of 16 (Figure 9). Therefore, selectivity is realized because this subpocket in BACE1 is responsive to increases in lipophilic bulk in this vector, whereas the same vector in CYP 2D6 directs towards the heme and has no impact on affinity. In addition, the ring fusion limits the requisite rotation of the thioamidine ring to optimally accommodate the aminomethyl substituent within the CYP 2D6 binding site. This discrepant response contrasts with the parallel responsiveness at BACE1 and CYP 2D6 for



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small changes in the benzylic amine of the monocyclic series, wherein equivalent increases in lipophilicity afforded corresponding enhancements in affinity for both BACE1 and CYP 2D6 receptors (6k vs 6u, 20x BACE1, 9x CYP 2D6). Future BACE1 design efforts focused on avoiding CYP 2D6 liability will therefore target manipulation of the amidine “head group” architecture, rather than optimization of P3 substituents.

Asp-228 S2’

Gly-230

Tyr-71

flap

S3

S1

10s loop

Figure 9. Co-crystal structure of 16 in BACE1. PDB ID: 5T1W Based upon its attractive balance of potency, selectivity, and CNS penetration, 16 was selected for further safety profiling. The compound showed excellent in vitro selectivity, as gauged by screening against the broad CEREP bioprint panel (

Aminomethyl-Derived Beta Secretase (BACE1

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